Quantification of cytokines, hormones, proteins, nucleic acids, and other such biomolecules contained in bodily fluids such as a blood plasma, a lymph fluid, and a tissue fluid, or detection of trace amounts of such biomolecules contained in bodily fluids is indispensable for early diagnosis of disease and accurate identification of disease conditions.
It is thought that serum protein concentrations are on the order of 10−16 to 10−12 M in cancers (NPL 1), neurological diseases (NPL 2), and in early stages of infection (NPL 3). Among the common techniques used for quantification or detection of biomolecules are ELISA, and bead assays using chemiluminescence and flow cytometry. However, these techniques have a detection sensitivity of at most about 10−12 M (NPL 4), and are very limited in terms of its applicability to early diagnosis of disease.
For example, assume a common blood test using an about 50-μl serum sample from a subject. Here, when the marker is 10−17 M (10 aM), only about 300 molecules would be present, and a technique that counts the molecules rather than measuring the concentration would be needed. As it currently stands, no such techniques are available for practical applications, and there is a need for a detection technique with an aM-order detection sensitivity.
PTL 1 discloses a method that uses flow cytometry to determine the number of microparticles that have captured antigens.
A recently proposed digital ELISA technique enables detection of a sample containing only 10 to 20 molecules per 100 μl (10−19 M concentration) by using antigen-antibody reaction (NPL 5). In this method, antibody-immobilized magnetic microparticles are used to capture antigens (detection target biomolecules), and are reacted with biotin-attached second antibodies, and then with streptavidin-attached galactosidase to introduce the enzyme onto the magnetic microparticles. The microparticles are then placed in an array of micro holes formed by etching the terminal of optical fiber bundles. This is acted upon by a chemiluminescence substrate, and the number of chemiluminescence spots is determined through the optical fibers to count the antigen molecules.